The present invention relates to polynucleotides derived from cDNA of a novel type of hepatitis C virus Korean type hepatitis C virus (KHCV), polypeptides encoded therein and antibodies directed against the polypeptides; and to diagnostics and vaccines employing any of these reagents, i.e., said polynucleotides, polypeptides and antibodies, as an active ingredient.
In general, virus-induced hepatitis has been known to be caused by various hepatitis viruses including hepatitis A virus, hepatitis B virus, hepatitis delta virus, hepatitis E virus, Cytomegalo virus and Esptein-Barr virus; and the genotypes of the viruses have been discovered since 1980, facilitating the development of diagnostics, vaccines and therapeutic agents.
Further, it has been discovered that a new type of hepatitis nicknamed as non-A non-B or C hepatitis, accounts for 80 to 90% of hepatitis caused by blood transfusion (Lancet, 2, 838-841 (1975)); and such post-transfusion hepatitis frequently progresses to cirrhosis or hepatocellular carcinoma up to about 50%.
The number of hepatitis C virus (HCV) present in patient""s blood is generally very small and the identity or specificity of the antigen and antibody systems associated with HCV has not been completely understood; and therefore, there have been many difficulties for developing therapeutic or diagnostic agents.
Consequently, the study on HCV has attracted a great deal of attention from numerous researchers (see, e.g., Alter, H. J. et al., Lancet, 459-463(1978); Tabor, E. et al., Lancet, 463-466 (1978); Hollinger, F. B. et al., Intervirology, 10, 60-68(1978); Wyke, R. J. et al., Lancet, 520-524(1979); Bradley, D. W. et al., J. Med. Virol., 9, 253-269(1979)).
Bradley et al., as discribed in Gastroenterology, 88, 773-779(1985), were able to determine the biochemical and biophysical characteristics of HCV by: infecting a champanzee with the serum of a hepatitis C patient; obtaining quantities of serum therefrom; extracting HCV from the serum; and analysing and studying HCV therewith.
Thereafter, many new studies were made with the HCV viruses isolated by employing the Bradley method for the development of agents to diagnose, prevent and/or treat hepatitis C.
Choo et al. cloned a partial cDNA fragment of HCV extracted from the serum of champanzee which had been infected with the serum of a hepatitis C patient; and proved that the protein produced by expressing the cDNA fragment in E. coli and yeast cell was immunologically reactive with the antibodies obtained from the serum of hepatitis C patients (Science 244, 359-362(1989).
Kuo et al. disclosed in Science, 244, 362-364(1989) that C100-3 protein prepared by expressing a partial HCV cDNA fragment, which was identified by Chiron Co. in U.S.A., fused with superoxide dismutase (SOD) gene in yeast was immunoreactive with the serum of hepatitis C patients and with 70% of the serum from those patients with post-transfusion hepatitis.
Further, Houghton et al. described the usefulness of HCV antigens, especially C100-3, encoded in HCV genomic sequence isolated from a champanzee contracted with hepatitis C (hereinafter, it is referred to as xe2x80x9cAmerican type HCVxe2x80x9d) for the preparation of vaccines and diagnostic agents capable of detecting anti-HCV antibodies (PCT WO 89/04669; WO 90/11089); and, established a diagnostic method employing enzyme immuno assay with said antigens, e.g., C100-3.
On the basis of the above invention, Ortho Diagnostic Systems Inc. of U.S.A. developed and distributed diagnostic agents for detecting anti-HCV antibodies in 1990. However, said C100-3 antigen used as the active ingredient for the diagnostic agents reacts only with the antibodies of patients with chronic hepatitis C, not with those of patients with acute hepatitis C especially during the early stage of the disease; and, further, it often exhibits false positive results due to the reaction of the fused protein, SOD (Shimizu, Y. K. et al., Proc. Natl. Acad. Sci. U.S.A., 87, 6441 (1990)).
On the other hand, partial HCV cDNA clones were prepared by employing the same method as of Houghton et al. from HCV taken from the serum collected from Japanese hepatitis C patients, including 5xe2x80x2-terminal region and structural genes encoding the core protein and the envelope protein; and the nucleotide sequence of the cDNA clones was determined from which it was discovered that the sequence is different from that of American type HCV about 10xcx9c15%, whereby the existence of a new type, what is called as Japaness type, of HCV was proven (Kubo, Y. et al., Nucl. Acid. Res., 17, 10367-10372(1989); Kato, N. et al., Proc. Japan. Acad., 65, 219-223(1990); Kaneko, S. et al., Lancet, 335, 976(1990); Takeuchi, K. et al., Gene, 91, 287-291(1990); Takenchi, K. et al., Nucl. Acid. Res., 18, 4626(1990); Takamizawa, A. et al., J. Virol., 65, 1105-1113(1991)); and, the specificity of the antigens derived from Japanese type HCV for preparing vaccines and diagnostic agents against Japanese type HCV was described by Okamoto, H. et al. in Japan. J. Exp. Med., 60, 167-177(1990).
Harada et al. further reported in J. Virol. 65, 3015 (1991) that when the core protein encoded in 5xe2x80x2-terminal portion of the structural gene was used for the antigen to diagnose anti-HCV antibodies which may be present in samples taken from putative patients, the antibodies could be detected 6 to 8 weeks earlier from the time of infection than the case of using C100-3 protein.
Lesniewski et al. also disclosed in Europen Patent Publication No. 725354 (1990) an improved diagnostic method using multiple antigens which was more sensitive and specific than the method of using C100-3 antigen alone; and Wang described in EP Publiction No. 442394 (1991) another diagnostic method wherein polypeptides consisting of 15xcx9c65 amino acids with epitope(s) selected from 10 different HCV epitopes were employed as antigens for detecting anti-HCV antibodies.
The above disclosures show that HCV diagnosis can be improved by empolying a mixture of polypeptides with different epitope(s) instead of using only one kind of antigen.
Furthermore, envelope proteins which exist on the surface of virus in the form of glycoproteins have been surfaced as a possible means for the development of vaccines as well as diagnostic agents. In the case of flavivirus which is very similar to HCV, it has been known that envelope proteins and non-structural protein 1(NS 1) play an important role in the induction of an immuno reaction of a host cell, and in binding itself to the receptors of host cell (F. Preugschart, J. Virol., 65, 4749-4758 (1991)). In addition, it has been reported that the formation of antibodies against envelope proteins is closely connected to recovery from hepatitis C (Lesniewski, R. et al., p 59; Watanabe et al., p 82, The 3rd International HCV Symposium, Strasbourg, France (1991)).
Further, Houghton et al. suggested the possibility that envelope 2(E2). Protein may prove to be an important antigen for preparing hepatitis C vaccines for the reason that said E2 protein is supposed to have a close relationship with immunoreaction mechanism since the amino terminal region of the E2 protein exhibits a conspicuous species heterogeneity (The 3rd International HCV Symposium, p 20, Strasbourg, France, 1991); and a comparison of the nucleotide sequences between the Japanese type HCV genome and the American type HCV genome has revealed that, while the nucleotide sequences encoding core proteins have a homology of about 91%, those encoding envelope proteins have a homology of about 74% (Takeuchi, K. et al., J. Gen. Vir., 71, 3027-3033(1990)).
As shown above, HCVs discovered in different countries may exhibit heterogeneity in various regions; and such heterogeneity may be a critical factor in deciding the effectiveness of vaccines and the sensitivity and accuracy of diagnostic agents.
Accordingly, the present invention pertains to the isolation and characterization of a novel type of HCV which is isolated from Korean hepatitis C patients (KHCV) and different from the already discovered HCVs including the American type and the Japanese type.
More specifically, the present invention provides a fully sequenced cDNA of said KHCV and partially sequenced cDNAs of several HCV varieties. Portions of the cDNA sequences derived from KHCV are useful as probes or primers to diagnose the presence of the virus in putative samples; and, diagnostic kits and methods utilizing such nucloeotide sequences also constitute further aspects of the invention.
In addition, the present invention provides polypeptides encoded in the above cDNA which are useful as reagents in diagnostic tests and/or as components of vaccines.
Said polypeptides encompass various polypeptides comprising a KHCV epitope including recombinant polypeptides such as fused polypeptides with a non-HCV protein; and purified forms thereof.
An additional aspect of the invention pertains to a recombinant expression vector comprising an open reading frame (ORF) of KHCV cDNA wherein said ORF is operably linked to a regulatory sequence compatible with a desired host organism and such vector may comprise: a nucleotide sequence encoding a non-KHCV protein for the preparation of a fused polypeptide of a polypeptide derived from KHCV and other type(s) of protein or polypeptide(s); a host cell transformed with the recombinant expression vector; and a polypeptide produced therefrom.
A further aspect of the present invention is a method for producing a polypeptide containing a KHCV epitope comprising: culturing host cells transformed with an expression vector containing a sequence encoding a polypeptide containing a KHCV epitope; and a polypeptide containing a KHCV epitope produced thereby.
Another aspect of the invention includes monoclonal antibody directed against a KHCV epitope.
A still additional aspect of the invention is directed to a hybridoma cell producing such monoclonal antibody.
Still further aspects of the invention are a diagnostic agent comprising one or more polypeptides which contain one or more KHCV epitopes as (an) active component(s) for detecting anti-KHCV antibodies in putative samples; and a diagnostic kit comprising such agent.
Still other aspects of the invention are a diagnostic agent comprising one or more monoclonal antibodies directed against the KHCV antigen to be detected as (an) active component(s) for dectecting HCV antigens in putative samples; and a diagnostic kit comprising such agent.
Even further aspect of the invention is a vaccine for the treatment and/or prevention of HCV infection comprising a polypeptide containing a KHCV epitope, and an inactivated or attenuated HCV.